I am using immunofluorescence on 10-micron thick tissue slices in order to see if two antigents are present in the same cell. The images clearly show two colors superimposing on each other. Do I need to perform confocal microscopy or other optical sectioning method to ascertain that both antigens colocalize in the same cell, or is the thickness of the tissue enough to rule out that the antigens are present in two different cells located in two different planes?Thanks.

This depends on the size of your cells. If they are large motor neurons, I would say you are on the save side, if they are for instance small lymphocytes you have to cut thinner sections. You can cut paraffin sections at 0.5 micrometer with a glass knive and an ultracut microtome. Then you can do the stainings on consecutive sections.

« Last Edit: August 28, 2007, 04:20:47 AM by dutchmaninvienna »

Logged

Jan Bauer, PhDAssociate ProfessorDiv. of NeuroimmunologyCenter for Brain ResearchMedical University of Vienna, Austria